沉默PHB减弱缺氧诱导的神经母细胞瘤自噬与增加顺铂化疗敏感性

Silencing PHB attenuated hypoxia-induced autophagy in neuroblastoma with greater sensitivity to cisplatin chemotherapy

目的探讨抗增殖蛋白(prohibitin,PHB)对缺氧诱导的神经母细胞瘤自噬及顺铂化疗敏感性的影响。方法体外培养SH-SY5Y和SK-N-SH细胞系,取对数期生长的细胞进行后续实验。分别转染sh-NC和sh-PHB到细胞中,采用RT-qPCR检测PHB的mRNA表达水平验证转染成功。缺氧处理后,分别用不同剂量(0、5μM、10μM、25μM、50μM和100μM)的顺铂处理细胞。CCK-8试剂盒检测各组细胞活性;Western blot检测细胞中Beclin1、LCⅡ/LCⅠ、Bax、Bcl-2和caspase-3的蛋白表达水平;流式细胞术检测细胞凋亡水平。采用SPSS 22.0软件包进行单因素方差分析或Student'st检验分析。结果和对照组比较,RT-qPCR和Western blot检测结果显示缺氧组中SH-SY5Y和SK-N-SH细胞中PHB的表达水平显著上调。转染sh-PHB后,RT-qPCR结果证实sh-PHB组中PHB的mRNA表达水平显著下调。Western blot结果显示和control组比较,Hypoxia组Beclin1的表达水平和LC3Ⅱ/LC3Ⅰ比值显著上调,然而和Hypoxia+sh-NC组比较,Hypoxia+sh-PHB组中Beclin1的表达水平和LC3Ⅱ/LC3Ⅰ比值显著下降。此外,CCK-8结果显示和Hypoxia+sh-NC组比较,Hypoxia+sh-PHB组中顺铂处理后细胞活性明显下降,提示顺铂敏感性增加。流式细胞术检测结果显示,和Hypoxia+sh-NC+Cisplatin组比较,Hypoxia+sh-PHB+Cisplatin组中凋亡水平显著增加。Western blot结果表明,和Hypoxia+sh-NC+Cisplatin组比较,Hypoxia+sh-PHB+Cisplatin组中caspase-3和Bax蛋白表达水平显著上调,Bcl-2蛋白表达水平下降。结论沉默PHB可以抑制缺氧诱导的神经母细胞瘤自噬,同时增加顺铂化疗敏感性。

Objective To explore the effect of PHB on autophagy and cisplatin sensitivity in hypoxia-induced neuroblastoma.Methods SH-SY5Y and SK-N-SH cells were cultured in vitro and logarithmically grown for subsequent experiments.Both sh-NC and sh-PHB were transfected into cells and successful transfection was verified by real-time quantitative polymerase chain reaction(RT-qPCR).After hypoxia treatment,cells were treated with different doses of cisplatin(0/5/10/25/50/100μM).CCK-8 kit was employed for detecting cell viability.The protein expression levels of Beclin1,LCII/LCI,Bax,Bcl-2 and caspase-3 in cells were detected by Western blot.And apoptosis was examined by flow cytometry.And SPSS 22.0 software package was utilized for one-way analysis of variance or Student's t test.And P<0.05 was deemed as statistically significant.Results As compared with control group,both RT-qPCR and Western blot indicated that the expression level of PHB was significantly up-regulated in hypoxia-induced SH-SY5Y/SK-N-SH cells.Successful transfection was confirmed by RT-qPCR.Western blot indicated that the expression level of Beclin1 and LC3II/LC3I ratio were significantly up-regulated in hypoxia group as compared with control group.However,the expression level of Beclin1 and LC3II/LC3I ratio declined markedly in hypoxia+sh-PHB group as compared with hypoxia+sh-NC group.In addition,CCK-8 assay indicated a marked decline of cell activity after cisplatin dosing in hypoxia+sh-PHB group versus hypoxia+sh-NC group.Flow cytometry hinted that apoptotic level spiked markedly in hypoxia+sh-PHB+cisplatin group as compared with hypoxia+sh-NC+cisplatin group.And Western blot revealed that the expression levels of caspase-3 and Bax were significantly up-regulated while the expression level of Bcl-2 dropped in hypoxia+sh-PHB+cisplatin group as compared with hypoxia+sh-NC+cisplatin group.Conclusions Silencing PHB may suppress hypoxia-induced autophagy and boost the sensitivity of cisplatin chemotherapy in neuroblastoma.